We propose to develop a rapid, low cost and high-throughput method for generating recombinant engineered antibodies with improved properties for proteomic and immunoassay applications. Engineered antibody constructs such as recombinant Fab fragments and chimeric antibodies will be generated and expressed by directly modifying the antibody genes in antibody-producing hybridoma cell lines, thereby eliminating the need for gene cloning and transfection. In this Phase I SBIR, we will demonstrate feasibility by modifying ten (10) hybridoma cell lines that produce antibodies used in MSD's commercial immunoassay kits. We will demonstrate that we can modify the cell lines so that they express their antibodies as recombinant Fab fragments or recombinant chimeras comprising Fab fragments linked to protein domains that enable site- specific labeling. The use of antibodies from established kits will make it straightforward to directly compare the assay performance of the new antibody constructs to the original full length antibodies. The long term goal of the program is to take advantage of beneficial properties of the chimeric constructs - lower non-specific interactions and the ability to control immobilization through site-specific labeling - to enable the development of high density arrays for proteomic applications. These improved multiplexed immunoassays will have significant value in both research and clinical diagnostics applications.